11/10/2016

1

Amylin(other names – islet amyloid polypeptide [IAPP], diabetes-associated peptide)

Professor Debbie L Hay

School of Biological Sciences

November 2016 AHS Scottsdale

Learning Objectives:

At the end of this presentation you should be able to -

• Define amylin activity

• Define amylin receptors

• Evaluate the importance of amylin receptors in CGRP activity

11/10/2016

2

Presentation outline

• Amylin: introduction and expression

• Amylin: glucoregulatory and satiety hormone

• Amylin: pain and other actions

• Amylin: receptors and relationship to CGRP receptors

o Amylin: receptor composition and pharmacology

o Amylin: receptor binding mechanisms

o Amylin: receptor expression – is AMY1 a CGRP or amylin receptor?

• Summary

Amylin: introduction and expression

4

11/10/2016

3

Amylin and CGRP are closely-related peptides

5

• Both 37 amino acids• ~40% identical in amino acid sequence

• Share important and highly conserved structural featureso N-terminal disulfideo C-terminal amide

• Some reported activities overlap• Some receptors overlap

Bower, R.L. & Hay, D.L., 2016 Brit J Pharmacol, 173(12):1883-98

Amylin expression

6

Rat hypothalamic slices

• Found in pancreatic islet β cells - co-secreted with insulin• Also found in stomach, hypothalamus and some neurons – Note: some “amylin” antibodies can also detect CGRP (see Tingstedt et al., 1999, J Histochem Cytochem)

Amylin

Tomita., 2003 Pathology. 35:34-36

Normal human islets

Li et al., 2015 Cell Metab. 22(6):1059-67

11/10/2016

4

Amylin expression – trigeminal ganglia (TG) neurons and perivascular fibres

7

Cat pial artery perivascular fibres

• Also found in some dorsal root ganglia (DRG) neurons

Amylin

Edvinsson et al., 2001 Sci World J. 1:168-80

Cat trigeminal ganglion

Amylin

Amylin expression – brainstem

8

Rat medulla oblongata

D’Este et al., 2000 Peptides. 1743-49

Spinal

trigeminal

tract

11/10/2016

5

Amylin: glucoregulatory and satiety hormone

9

Amylin – physiological role as a glucoregulatory hormone versus pathological role in islet amyloid

10

Islet amyloid

Westermark et al., 2011 Physiol. Rev. 91:795-826

Bower, R.L. & Hay, D.L., 2016 Brit J Pharmacol, 173(12):1883-98

Amyloidogenic region

• Amylin can form amyloid under some circumstances

11/10/2016

6

Amylin – physiological role

11Hay, D. L. et al., 2015 Pharmacol. Rev. 67(3):564-600

• Co-secreted with insulin from pancreas in response to nutrient intake

• Has a complementary role to insulin in controlling blood glucose

• Deficient in type I and late-stage type II diabetes, where there is β-cell loss

NH2

Amylin analogue is an approved therapeutic agent in diabetes

12

www.symlin.com

NH2

NH2

Human amylin Rat amylin

Pramlintide

NH2

FDA approved

FDA approved

In trials

Type I diabetes;

insulin adjunct

Insulin-requiring

type II diabetes;

insulin adjunct

Obesity

Bower, R.L. & Hay, D.L., 2016 Brit J Pharmacol, 173(12):1883-98

Hay, D. L. et al., 2015 Pharmacol. Rev. 67(3):564-600

11/10/2016

7

Pramlintide reduces food intake and increases “fullness”

13

Energy and macronutrient intake

at an ad libitum buffet mealFullness scores

Chapman et al., 2005 Diabetalogia 48(5):838-48

Single s.c. injection of placebo or 120 μg of pramlintide

in 11 subjects with insulin-treated type 2 diabetes

Placebo (circles)

Pramlintide (squares)

Amylin complements the actions of other metabolic hormones to produce weight loss

14

Mechanisms for leptin and amylin co-agonism in the

control of body weight and glucose homeostasis

Sadry, S. A. & Drucker, D. J., 2013 Nat. Rev. Endocrinol. 9(7):425-33 Roth et al., 2008 Proc Natl Acad Sci USA 105:7257-7262

Human clinical trial of pramlintide in obesity

11/10/2016

8

Side effects associated with pramlintide use in humans

15

Younk et al., 2011 Expert Opin Pharmacother 12(9):1439-1451

• Tested in normal human subjects, T1D and T2D, no specific headache cohort

• Severe hypoglycemia“In a dose‐rising tolerability

study in non‐diabetic

volunteers, the highest

doses were 10 mg, ∼80‐ to

300‐fold higher than

anti‐diabetic doses.

Dose‐limiting side effects

were nausea and vomiting

at 5 mg and 10 mg doses,

with no effects reported at

doses of 0.3, 1, and 3 mg

(Moyseset al., 1993).” From

Young, 1995 Adv. Pharmacol. 52:289-320

Pramlintide and headache?

16

Heise et al., 2004 Metabolism 53(9):1227-1232

• “Treatment with a primed, continuous IV infusion of pramlintide (∼16 µg/h) produced consistent mean plasma pramlintide concentrations of 83 ± 3.6, 82 ± 3.7, and 74 ± 3.8 pmol/L”

• 18 “healthy” subjects• “Back pain, headache, and diarrhea were the 3 most common adverse events accounting for 11% versus 0%, 17% versus 6%, and 11% versus 6% in the pramlintide versus placebo groups, respectively. Those adverse events reported were of mild-to-moderate intensity.”

PubMed search terms: “amylin headache”, “amylin migraine”, “pramlintideheadache”, “pramlintide migraine”, “symlin headache”, “symlin migraine”

11/10/2016

9

Amylin: pain and other actions

17

Amylin and pain

18

• Overall amylin appears to be anti-nociceptive in pharmacology studies (e.g. reduced pain in formalin and acetic acid tests)

• Amylin knockout has anti-nociceptive phenotype

• Calcitonin is anti-nociceptive

Gebre-Medhin et al., 1998 Brain Res Mol Brain Res 63(1):180-3

11/10/2016

10

Other activities of amylin

19

From: Hay, D. L. et al., 2015

Pharmacol. Rev. 67(3):564-600

Amylin: receptors and relationship to CGRP receptors

20

11/10/2016

11

Amylin: receptor composition and pharmacology

21

Amylin and CGRP receptors

22

• G protein-coupled receptors (class B)• For summary see: www.guidetopharmacology.org• Have different subunits comprising a 7-transmembrane GPCR and a 1-transmembrane accessory proteino Calcitonin receptor-like receptor (CLR) - GPCRo Calcitonin receptor (CTR) - GPCRo Receptor activity-modifying proteins 1, 2 and 3 – accessory proteino These combine together to form different subtypes of receptors for the calcitonin peptide family – CGRP, amylin, calcitonin, adrenomedullin, adrenomedullin 2

11/10/2016

12

Amylin receptors – composition

23

Calcitonin receptor

CTR

AMY1 receptor

CTR + RAMP1

AMY2 receptor

CTR + RAMP2

AMY3 receptor

CTR + RAMP3

Increased amylin affinity, compared to CTR

CGRP and adrenomedullin receptors are closely-related to amylin receptors

24

CGRP receptor

CLR + RAMP1

AM1 receptor

CLR + RAMP2

AM2 receptor

CLR + RAMP3

• Note: the CGRP receptor and AMY1 receptor share RAMP1

11/10/2016

13

There is considerable overlap in pharmacology between receptors

25

AMY2 receptor

CTR + RAMP2

AMY3 receptor

CTR + RAMP3AM2 receptor

CLR + RAMP3

CGRP Amylin

Calcitonin receptor

CTRCGRP receptor

CLR + RAMP1

AM1 receptor

CLR + RAMP2

AMY1 receptor

CTR + RAMP1

CGRP8-37 AC187Olcegepant

Example agonists

and their overlapping

activities

Example antagonists

and their overlapping

activities

CGRP receptor antagonists can fully inhibit CGRP activity at AMY1 receptors

Hay & Walker, 2016 Headache, submitted

CGRP receptor

CLR + RAMP1AMY1 receptor

CTR + RAMP1

11/10/2016

14

Amylin but not CGRP is resistant to olcegepant (BIBN4096) antagonism at the AMY1 receptor

Walker, et al., 2016 Cephalalgia, in revision

log[h CGRP] (M)

0

25

50

75

100

125

-12 -11 -10 -9 -8 -7 -6

h CGRP

+ 1 M Olcegepant

0

log[hAmylin] (M)cAMP (% Maxim

um)

0

25

50

75

100

125

-12 -11 -10 -9 -8 -7 -6

hAmylin

+ 1 M Olcegepant

0

pA2 7.88 (13.2 nM) pA2 6.72 (190 nM)

Olcegepant (BIBN4096) antagonism at the AMY1 receptor depends on the measured activity, further reducing selectivity

Walker, et al., 2016 Cephalalgia, in revision

Rat TG neuronsHuman

CGRP

Human

AMY1

Antagonist

selectivity

CGRP vs. AMY1

αCGRP vs. Olcegepant

cAMP7.79 ± 0.21 (6)

[16.2 nM]

10.00 ± 0.20 (5)

[0.1 nM]

7.88 ± 0.12 (5)

[13.2 nM]

132

pCREB9.09 ± 0.04 (4)*

[0.8 nM]

10.00 ± 0.24 (6)

[0.1 nM]

8.58 ± 0.15 (9)*

[2.6 nM]

26

pp388.51 ± 0.39 (5)

[3.1 nM]N.D. N.D.

-_

αCGRP vs. Telcagepant

cAMP5.72 ± 0.14 (4)

[1.9 µM]

8.92 ± 0.05 (5)

[1.2 nM]

7.37 ± 0.12 (5)

[42.7 nM]

35

pCREB N.D.8.71 ± 0.04 (5)

[1.9 nM]

7.69 ± 0.32 (5)

[20.4 nM]

10

AC

[cAMP][cAMP]

βγβγαsαs

CGRP

pCREB

response

11/10/2016

15

Amylin: receptor binding mechanisms

29

Amylin – structural motifs and receptor binding mechanism

30

KC

N

T A

T

C A

T

Q

R

L

A

N

F

L

S

SN

NFILS

ST

NV

V

H

GA

G

SN

T Y NH2

N terminal

activation loop α-helix

C terminal affinity

anchor

Mid-region

Human amylin

2L86

Solution NMR structure of

human amylin in SDS

micelles at pH 7.3

TM region

ECD

AMY1 receptor

CTR + RAMP1

Nanga, R.B. et al., 2011 Biochem. Biophys. Acta 1808:2337-2342

C-terminal amide

11/10/2016

16

Summary of human amylin alanine scan data

31

Human KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

Rat KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY

Mouse KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY

Baboon ICNTATCATQRLANFLVRSSNNFGTILSSTNVGSDTY

Macaque KCNTATCATQRLANFLVRSSNNFGTILSSTNVGSDTY

Bear KCNTATCATQRLANFLVRSGNNLGAILSPTNVGSNTY

Bovine KCGTATCETQRLANFLAPSSNKLGAIFSPTKMGSNTY

Porcine KCNMATCATQHLANFLDRSRNNLGTIFSPTKVGSNTY

Dog KCNTATCATQRLANFLVRTSNNLGAILSPTNVGSNTY

Cat KCNTATCATQRLANFLIRSSNNLGAILSPTNVGSNTY

Ferret KCNTATCVTQRLANFLIHSSNNLGAILLPTDVGSNTY

Goldfish KCNTATCVTQRLADFLVRSSNTRGTVYAPTNVGANTY

No effect

>10-fold decrease EC50

<10<10--fold decrease ECfold decrease EC5050

Increase ECIncrease EC5050

Cysteine Cysteine –– not mutatednot mutated

Bower, R.L. & Hay, D.L., 2016 Brit J Pharmacol, 173(12):1883-98

Bower, Yule et al., unpublished

A shared CGRP binding site in the AMY1 and CGRP receptor extracellular domains?

32Gingell et al., 2016 Cell Discovery 2:16012

Booe et al., 2015 Mol. Cell. 58(6):1040-52

CGRP receptor

CLR + RAMP1, CGRPAMY1 receptor

CTR + RAMP1 , CGRP

11/10/2016

17

Small molecule antagonist binding to the CGRP receptor uses W74 in RAMP1, which is shared with AMY1

33 Archbold et al., 2011 Trends Pharmacol, Sci. 32(10):591-600

Olcegepant uses the same key RAMP1 residue to bind both AMY1and CGRP receptors

34

WT A

MY1(a) AMY

W74A AMY

1(a) AMY

W74K AM

Y1(a) AM

Y

WT AMY

1(a)CG

RP

W74A

AMY

1(a)CG

RP

W74K AM

Y1(a)

CGRP

5

6

7

8

9

10

11

***

**

WT C

GRP

CGRP

W74A CGR

P CG

RP

W74K

CGR

P CG

RP

WT CGR

P CG

RP

W74A CG

RP C

GRP

W74K CG

RP C

GRP

5

6

7

8

9

10

11

*** ****

Data replotted from Hay et al., 2006 Mol Pharmacol, 70(6), 1984-1991

11/10/2016

18

Amylin: receptor expression – is AMY1 a CGRP or amylin receptor?

35

Tissue expression of amylin receptors

36

• Largely unknown

• Amylin binding shows widespread expression in the brain with some CGRP overlap

• mRNA shows multiple RAMPs co-expressed

• Pharmacological tools cannot distinguish receptors

• Limited CTR/RAMP1 antibody data; no reliable RAMP2 or RAMP3 antibodies

• CTR splice variants (also CGRP-responsive e.g. Qi et al., 2013 Brit. J. Pharmacol. 168:644-

657)

• Not known which receptor(s) mediate amylin action in vivo and are the target for pramlintide

• Evidence for AMY1 as an amylin receptor vs CGRP receptor?

Van Rossum et al., 1995 Can. J. Pysiol. Pharmacol. 73(7):1037-41

11/10/2016

19

Summary of amylin-related transgenic and knockout models

37

Hay, D. L. et al., 2015 Pharmacol. Rev. 67(3):564-600

RAMPs affect the activity of other receptors

38Hay & Pioszak 2016, Ann. Rev. Pharmacol. Toxicol. 56:469-87

11/10/2016

20

CTR expression in the brain

39

9B4 mAb, HEK293S cells

Walker, C.S. et al., 2015 Ann. Clin. Transl. Neurol. 2(6):595-608

Hay, D. L. et al., 2015 Pharmacol. Rev. 67(3):564-600

Brainstem

Core subunit:

Calcitonin receptor

CTR

CTR in human brainstem

40 Bower, R.L. et al,. 2016 Am. J. Physiol. – Reg. Int. Comp.Physiol. 310(9):R788-93

NTS = nucleus of the solitary tract

AP = area postrema

Nissl

8 human cases, 65-82 yr, no neurological disease

CTR- 9B4

Walker, C.S. et al., 2015 Ann. Clin. Transl. Neurol. 2(6):595-608

11/10/2016

21

Functional AMY1 receptors in TG and brainstem but where else?

41

The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

• CTR + RAMP1 are co-expressed in rat and human TG and human brainstem

• Receptor signals in TG

Walker et al., Annals of Clinical and Translational Neurology, 2(6): 595–608, 2015

0

20

40

60

80

100

120

-11 -10 -9 -8 -7 -6 -50

r CGRP

+1 M AC187

log[r CGRP] (M)

Rat TG

neurons

Human

brainstemRat TG

• Amylin is natural glucoregulatory hormone that controls satiety• Amylin and CGRP have overlapping sequences, structures, effects and receptors

• Amylin and CGRP have complex multi-subunit receptors• The AMY1 receptor is a CGRP receptor• CGRP could act through this receptor in migraine• Many “CGRP receptor” antagonists are also AMY1 antagonists• The role of CGRP and AMY1 receptors in CGRP action needs further defining to inform the safety and efficacy of anti-CGRP classes of drug

Summary

42